Methods and reagents for decreasing clinical reaction to allergy

ABSTRACT

It has been determined that allergens, which are characterized by both humoral (IgE) and cellular (T cell) binding sites, can be modified to be less allergenic by modifying the IgE binding sites. The IgE binding sites can be converted to non-IgE binding sites by masking the site with a compound that prevents IgE binding or by altering as little as a single amino acid within the protein, most typically a hydrophobic residue towards the center of the IgE-binding epitope, to eliminate IgE binding. The method allows the protein to be altered as minimally as possible, other than-within the IgE-binding sites, while retaining the ability of the protein to activate T cells, and, in some embodiments by not significantly altering or decreasing IgG binding capacity The examples use peanut allergens to demonstrate alteration of IgE binding sites. The critical amino acids within each of the IgE binding epitopes of the peanut protein that are important to immunoglobulin binding have been determined. Substitution of even a single amino acid within each of the epitopes led to loss of IgE binding. Although the epitopes shared no common amino acid sequence motif, the hydrophobic residues located in the center of the epitope appeared to be most critical to IgE binding.

The present application is a continuation application of, and claimspriority under 35 U.S.C. § 120 to, co-pending U.S. patent applicationSer. No. 09/478,668, filed Jan. 6, 2000 (the '668 application).

The present application further claims priority under 35 U.S.C. § 120 toU.S. patent application Ser. No. 09/141,220, filed Aug. 27, 1998 andcurrently pending (the '220 application), which was co-pending with, andshared at least one inventor in common with, the '668 application. The'668 application was a divisional of the '220 application.

The present application further claims priority under 35 U.S.C. § 120 toU.S. patent application Ser. No. 09/106,872, filed Jun. 29, 1998 and nowissued as U.S. Pat. No. 6,486,311 (the '872 application), which wasco-pending with, and shared at least one inventor in common with, the'220 application. The '220 application was a continuation-in-part of the'872 application.

The present application also claims priority under 35 U.S.C. § 119(e)to; U.S. Provisional Patent Application No. 60/077,763, filed Mar. 13,1998; U.S. Provisional Patent Application No. 60/074,590 filed on Feb.13, 1998; U.S. Provisional Patent Application No. 60/074,624 filed onFeb. 13, 1998; U.S. Provisional Patent Application No. 60/074,633 filedon Feb. 13, 1998; and to U.S. Provisional Patent Application No.60/073,283, filed Jan. 31, 1998. Each of these Provisional PatentApplications is currently expired, but was co-pending with and filed byat least one common inventor as the '872 application.

Each of the above applications to which priority is claimed is herebyincorporated by reference in its entirety.

The United States government has rights in this invention by virtue ofgrants from the National Institute of Health RO1-AI33596.

BACKGROUND OF THE INVENTION

Allergic disease is a common health problem affecting humans andcompanion animals (mainly dogs and cats) alike. Allergies exist tofoods, molds, grasses, trees, insects, pets, fleas, ticks and othersubstances present in the environment. It is estimated that up to 8% ofyoung children and 2% of adults have allergic reactions just to foodsalone. Some allergic reactions (especially those to foods and insects)can be so severe as to be life threatening. Problems in animals tend tobe less severe, but very common. For example, many dogs and cats haveallergies to flea saliva proteins, grasses, and other common substancespresent in the environment.

Allergy is manifested by the release of histamines and other mediatorsof inflammation by mast cells which are triggered into action when IgEantibodies bound to their receptors on the mast cell surface are crosslinked by antigen. Other than avoidance, and drugs (e.g.,antihistamines, decongestants, and steroids) that only treat symptomsand can have unfortunate side effects and often only provide temporaryrelief, the only currently medically accepted treatment for allergies isimmunotherapy. Immunotherapy involves the repeated injection of allergenextracts, over a period of years, to desensitize a patient to theallergen. Unfortunately, traditional immunotherapy is time consuming,usually involving years of treatment, and often fails to achieve itsgoal of desensitizing the patient to the allergen. Furthermore, it isnot the recommended treatment for food allergies, such as peanutallergies, due to the risk of anaphylaxis.

Noon (Noon, Lancet 1911; 1:1572-73) first introduced allergen injectionimmunotherapy in 1911, a practice based primarily on empiricism withnon-standardized extracts of variable quality. More recently theintroduction of standardized extracts has made it possible to increasethe efficacy of immunotherapy, and double-blind placebo-controlledtrials have demonstrated the efficacy of this form of therapy inallergic rhinitis, asthma and bee-sting hypersensitivity (BSAC WorkingParty, Clin. Exp. Allergy 1993; 23:1-44). However, increased risk ofanaphylactic reactions has accompanied this increased efficacy. Forexample, initial trials of immunotherapy to food allergens hasdemonstrated an unacceptable safety: efficacy ratio (Oppenheimer et al.J. Allergy Clin. Immun. 1992; 90:256-62; Sampson, J. Allergy Clin.Immun. 1992; 90:151-52; Nelson et al. J. Allergy Clin. Immun. 1996;99:744-751). Results like these have prompted investigators to seekalternative forms of immunotherapy as well as to seek other forms oftreatment.

Initial trials with allergen-non-specific anti-IgE antibodies to depletethe patient of allergen-specific IgE antibodies have shown early promise(Boulet, et al. 1997; 155:1835-1840; Fahy, et al. American J Respir.Crit. Care Med. 1997; 155:1828-1834; Demoly P. and Bousquet J. AmericanJ Resp. Crit. Care Med. 1997; 155:1825-1827). On the other hand, trialsutilizing immunogenic peptides (representing T cell epitopes) have beendisappointing (Norman, et al. J. Aller. Clin. Immunol. 1997; 99:S127).Another form of allergen-specific immunotherapy which utilizes injectionof plasmid DNA (Raz et al. Proc. Nat. Acad. Sci. USA 1994; 91:9519-9523;Hz et al. Int. Immunol. 1996; 8:1405-1411) remains unproven.

There remains a need for a safe and efficacious therapy for allergies,especially those where traditional immunotherapy is ill advised due torisk to the patient or lack of efficacy. There is also a need foralternatives to therapies, for example, by creating foods, materials orsubstances that do not include the allergens that are most problematic,or which contain modified allergens which do not elicit the samereaction. While the technology to make genetically engineered plants andanimals is at this point well established, useful modifications wouldrequire understanding how allergens can be modified so that they retainthe essential functions for the plants' and animals' nutritional value,taste characteristics, etc., but no longer elicit as severe an allergicresponse.

It is therefore an object of the present invention to provide a methodfor decreasing the allergenicity of allergens either by modifying theallergen itself or by producing a compound that would mask the epitopeand thus prevent binding of IgE.

It is a further object of the present invention to provide allergensthat elicit fewer IgE mediated responses.

It is still another object of the present invention to provide a methodto make genetically engineered plants and animals that elicit less of anallergic response than the naturally occurring organisms.

SUMMARY OF THE INVENTION

It has been determined that allergens, which are characterized by bothhurnoral (IgG and IgE) and cellular (T cell) binding sites, can be madeless allergenic by modifying the IgE binding sites. The IgE bindingsites can be eliminated by masking the site with a compound that wouldprevent IgE binding or by altering as little as a single amino acidwithin the protein to eliminate IgE binding. The method allows theprotein to be altered as minimally as possible, (i.e. only within theIgE-binding sites) while retaining the ability of the protein toactivate T cells and, optionally, to bind IgG. Binding sites areidentified using known techniques, such as by binding with antibodies inpooled sera obtained from individuals known to be immunoreactive withthe allergen to be modified. Proteins that are modified to alter IgEbinding are screened for binding with IgG and/or activation of T cells.

Peanut allergens (Ara h 1, Ara h 2, and Ara h 3) have been used in theexamples to demonstrate alteration of IgE binding sites while retainingbinding to IgG and activation of T cells. The critical amino acidswithin each of the IgE binding epitopes of the peanut protein that areimportant to immunoglobulin binding were determined. Substitution ofeven a single amino acid within each of the epitopes led to loss of IgEbinding. Although the epitopes shared no common amino acid sequencemotif, the hydrophobic residues located in the center of the epitopeappeared to be most critical to IgE binding.

Standard techniques such as a skin test for wheal and flare formationcan be used to assess decreased allergenicity of modified proteins,created as described in the examples. The modified allergens can also betested for binding to IgG and proliferation of T cells, and modifiedallergens selected for optimal stimulation of T cells and binding IgG.

The immunotherapeutics can be delivered by standard techniques, usinginjection, by aerosol, sublingually, topically (including to a mucosalsurface), and by gene therapy (for example, by injection of the geneencoding the inimunotherapeutic into muscle or skin where it istransiently expressed for a time sufficient to induce tolerance).

This method and the criteria for identifying and altering allergens canbe used to design useful proteins (including nucleotide moleculesencoding the proteins) for use in immunotherapy, to make a vaccine andto genetically engineer organisms such as plants and animals which thenproduce proteins with less likelihood of eliciting an IgE response.Techniques for engineering plants and animals are well known. Based onthe information obtained using the method described in the examples, onecan engineer plants or animals to cause either site specific mutationsin the gene encoding the protein(s) of interest, or to knock out thegene and then insert the gene encoding the modified protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an example of how IgE binding epitopes were mapped to aspecific amino acid sequence on the Ara h 1 allergen.

FIG. 2 shows how IgE binding epitopes were mapped to a specific aminoacid sequence on the Ara h 2 allergen.

FIG. 3 shows how IgE binding epitopes were mapped to a specific aminoacid sequence on the Ara h 3 allergen.

FIG. 4 shows how amino acids critical to IgE binding were identified.

FIG. 5A shows the location of altered residues within the Ara h 2 aminoacid sequence (SEQ ID NO 4).

FIG. 5B shows the effect the modified Ara h 2 protein has on IgEbinding.

FIG. 5C shows the effect the modified Ara h 2 protein has on IgGbinding.

FIG. 6 shows the results of T-cell proliferation assays using thewild-type and modified Ara h 2 protein.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The following definitions are used herein.

An antigen is a molecule that elicits production of antibody (a humoralresponse) or an antigen-specific reaction with T cells (a cellularresponse).

An allergen is a subset of antigens which elicits IgE production inaddition to other isotypes of antibodies.

An allergic reaction is one that is IgE mediated with clinical symptomsprimarily involving the cutaneous (uticaria, angiodema, pruritus),respiratory (wheezing, coughing, laryngeal edema, rhinorrhea,watery/itching eyes), gastrointestinal (vomiting, abdominal pain,diarrhea), and cardiovascular (if a systemic reaction occurs) systems.

An epitope is a binding site including an amino acid motif of betweenapproximately six and fifteen amino acids which can be bound by eitheran immunoglobulin or recognized by a T cell receptor when presented byan antigen presenting cell in conjunction with the majorhistocompatibility complex (MHC). A linear epitope is one where theamino acids are recognized in the context of a simple linear sequence. Aconformational epitope is one where the amino acids are recognized inthe context of a particular three dimensional structure.

An immunodominant epitope is one which is bound by antibody in a largepercentage of the sensitized population or where the titer of theantibody is high, relative to the percentage or titer of antibodyreaction to other epitopes present in the same protein.

A decreased allergic reaction is characterized by a decrease in clinicalsymptoms following treatment of symptoms associated with exposure to anallergen, which can involve respiratory, gastrointestinal, skin, eyes,ears and mucosal surfaces in general.

An antigen presenting cell (an APC) is a cell which processes andpresents peptides to T cells to elicit an antigen-specific response.

Immunostimulatory sequences are oligodeoxynucleotides of bacterial,viral or invertebrate origin that are taken-up by APCs and activate themto express certain membrane receptors (e.g., B7-1 and B7-2) and secretevarious cytokines (e.g., IL-1, IL-6, IL-12, TNF). Theseoligodeoxynucleotides containing unmethylated CpG motifs cause briskactivation and when injected into animals in conjunction with antigen,appear to skew the immune response to a Th1-type response. See, forexample, Yamamoto, et al., Microbiol. Immunol. 36, 983 (1992); Krieg, etal., Nature 374, 546-548 (1995); Pisetsky, Immunity 5, 303 (1996); andZimmerman, et al., J. Immunol. 160,3627-3630 (1998).

I. Diagnostic and Therapeutic Reagents

The first step in making the modified allergen is to identify IgEbinding sites and/or immunodominant IgE binding sites. The second stepis to mutate one or more of the IgE binding sites, preferably includingat a minimum one of the immunodominant sites, or to react the allergenwith a compound that selectively blocks binding to one or more of theIgE binding sites. The third step is to make sufficient amounts of themodified allergen for administration to persons or animals in need oftolerance to the allergen, where the modified allergen is administeredin a dosage and for a time to induce tolerance, or for diagnosticpurposes. The modified allergen can be administered by injection, or insome cases, by ingestion or inhalation.

A. Allergens

Many allergens are known that elicit allergic responses, which may rangein severity from mildly irritating to life-threatening. Food allergiesare mediated through the interaction of IgE to specific proteinscontained within the food. Examples of common food allergens includeproteins from peanuts, milk, grains such as wheat and barley, soybeans,eggs, fish, crustaceans, and mollusks. These account for greater than90% of the food allergies (Taylor, Food Techn. 39, 146-152 (1992). TheIgE binding epitopes from the major allergens of cow milk (Ball, et al.(1994) Clin. Exp. Allergy, 24, 758-764), egg (Cooke, S. K. and Sampson,H. R. (1997) J. Immunol., 159, 2026-2032), codfish (Aas, K., andElsayed, S. (1975) Dev. Biol. Stand. 29, 90-98), hazel nut (Elsayed, etal. (1989) Int. Arch. Allergy Appl. Immunol. 89, 410-415), peanut (Burkset al., (1997) Eur. J. Biochemistry, 245:334-339; Stanley et al., (1997)Archives of Biochemistry and Biophysics, 342:244-253), soybean (Herein,et al. (1990) Int. Arch. Allergy Appl. Immunol. 92, 193-198) and shrimp(Shanty, et al. (1993) J. Immunol. 151, 5354-5363) have all beenelucidated, as have others. Other allergens include proteins frominsects such as flea, tick, mite, fire ant, cockroach, and bee as wellas molds, dust, grasses, trees, weeds, and proteins from mammalsincluding horses, dogs, cats, etc.

The majority of allergens discussed above elicit a reaction wheningested, inhaled, or injected. Allergens can also elicit a reactionbased solely on contact with the skin. Latex is a well known example.Latex products are manufactured from a milky fluid derived from therubber tree, Hevea brasiliensis and other processing chemicals. A numberof the proteins in latex can cause a range of allergic reactions. Manyproducts contain latex, such as medical supplies and personal protectiveequipment. Three types of reactions can occur in persons sensitive tolatex: irritant contact dermatitis, and immediate systemichypersensitivity. Additionally, the proteins responsible for theallergic reactions can fasten to the powder of latex gloves. This powdercan be inhaled, causing exposure through the lungs. Proteins found inlatex that interact with IgE antibodies were characterized bytwo-dimensional electrophoresis. Protein fractions of 56, 45, 30, 20,14, and less than 6.5 kd were detected (Posch A. et al., (1997) J.Allergy Clin. Immunol. 99(3), 385-395 ). Acidic proteins in the 8-14 kdand 22-24 kd range that reacted with IgE antibodies were also identified(Posch A. et al., (1997) J. Allergy Clin. Immunol. 99(3), 385-395. Theproteins prohevein and hevein, from hevea brasiliensis, are known to bemajor latex allergens and to interact with IgE (Alenius, H., et al.,Clin. Exp. Allergy 25(7), 659-665; Chen Z., et al., (1997) J. AllergyClin. Immunol. 99(3), 402-409). Most of the IgE binding domains havebeen shown to be in the hevein domain rather than the domain specificfor prohevein (Chen Z., et al., (1997) J. Allergy Clin. Immunol. 99(3),402-409). The main IgE-binding epitope of prohevein is thought to be inthe N-terminal, 43 amino acid fragment (Alenius H., et al., (1996) J.Immunol. 156(4), 1618-1625). The hevein lectin family of proteins hasbeen shown to have homology with potato lectin and snake venomdisintegrins (platelet aggregation inhibitors) (Kielisqewski, M. L., etal., (1994) Plant J. 5(6), 849-861).

B. Identification of IgE Binding Sites

Allergens typically have both IgE and IgG binding sites and arerecognized by T cells. The binding sites can be determined either byusing phage display libraries to identify conformational epitopes(Eichler and Houghten, (1995) Molecular Medicine Today 1, 174-180;Jensen-Jarolim et al., (1997) J. Appl. Clin. Immunol. 101, 5153a) or byusing defined peptides derived from the known amino acid sequence of anallergen (see examples below), or by binding of whole protein or proteinfragments to antibodies, typically antibodies obtained from a pooledpatient population known to be allergic to the allergen. It is desirableto modify allergens to diminish binding to IgE while retaining theirability to activate T cells and in some embodiments by not significantlyaltering or decreasing IgG binding capacity. This requires modificationof one or more IgE binding sites in the allergen.

A preferred modified allergen is one that can be used with a majority ofpatients having a particular allergy. Use of pooled sera from allergicpatients allows determination of one or more immunodominant epitopes inthe allergen. Once some or all of the IgE binding sites are known, it ispossible to modify the gene encoding the allergen, using site directedmutagenesis by any of a number of techniques, to produce a modifiedallergen as described below, and thereby express modified allergens. Itis also possible to react the allergen with a compound that achieves thesame result as the selective mutation, by making the IgE binding sitesinaccessible, but not preventing the modified allergen from activating Tcells, and, in some embodiments, by not significantly altering ordecreasing IgG binding.

Assays to assess an immunologic change after the administration of themodified allergen are known to those skilled in the art. Conventionalassays include RAST (Sampson and Albergo, 1984), ELISAs (Burks, et al.1986) immunoblotting (Burks, et al. 1988), and in vivo skin tests(Sampson and Albergo 1984). Objective clinical symptoms can be monitoredbefore and after the administration of the modified allergen todetermine any change in the clinical symptoms.

It may be of value to identify IgEs which interact with conformationalrather than linear epitopes. Due to the complexity and heterogeneity ofpatient serum, it may be difficult to employ a standard immobilizedallergen affinity-based approach to directly isolate these IgEs inquantities sufficient to permit their characterization. These problemscan be avoided by isolating some or all of the IgEs which interact withconformational epitopes from a combinatorial IgE phage display library.

Steinberger et al. (Steinberger, P., Kraft D. and Valenta R. (1996)“Construction of a combinatorial IgE library from an allergic patient:Isolation and characterization of human IgE Fabs with specificity forthe major Timothy Grass pollen antigen,” Phl p. 5 J. Biol. Chem. 271,10967-10972) prepared a combinatorial IgE phage display library frommRNA isolated from the peripheral blood mononuclear cells of a grassallergic patient. Allergen-specific IgEs were selected by panningfilamentous phage expressing IgE Fabs on their surfaces against allergenimmobilized on the wells of 96 well microtiter plates. The cDNAs werethan isolated from allergen-binding phage and transformed into E colifor the production of large quantities of monoclonal, recombinant,allergen-specific IgE Fabs.

If native allergen or full length recombinant allergen is used in thepanning step to isolate phage, then Fabs corresponding to IgEs specificfor conformational epitopes should be included among theallergen-specific clones identified. By screening the individualrecombinant IgE Fabs against denatured antigen or against the relevantlinear epitopes identified for a given antigen, the subset ofconformation-specific clones which do not bind to linear epitopes can bedefined.

To determine whether the library screening has yielded a completeinventory of the allergen-specific IgEs present in patient serum, animmunocompetition assay can be performed. Pooled recombinant Fabs wouldbe preincubated with immobilized allergen. After washing to removeunbound Fab, the immobilized allergen would then be incubated withpatient serum. After washing to remove unbound serum proteins, anincubation with a reporter-coupled secondary antibody specific for IgEFe domain would be performed. Detection of bound reporter would allowquantitation of the extent to which serum IgE was prevented from bindingto allergen by recombinant Fab. Maximal, uncompeted serum IgE bindingwould be determined using allergen which had not been preincubated withFab or had been incubated with nonsense Fab. If IgE binding persists inthe face of competition from the complete set of allergen-specific IgEFab clones, this experiment can be repeated using denatured antigen todetermine whether the epitopes not represented among the cloned Fabs arelinear or conformational.

Production of Recombinant or Modified Allergens

A modified allergen will typically be made using recombinant techniques.Expression in a procaryotic or eucaryotic host including bacteria,yeast, and baculovirus-insect cell systems are typically used to producelarge (mg) quantities of the modified allergen. It is also possible tomake the allergen synthetically, if the allergen is not too large, forexample, less than about 25-40 amino acids in length.

Production of Transzenic Plants and Animals

Transgenic plants or animals expressing the modified allergens have twopurposes. First, they can be used as a source of modified allergen foruse in immunotherapy and second, appropriately modified plants oranimals can be substituted for the original plant or animal, makingimmunotherapy unnecessary. Furthermore, it is possible that eatingmodified peanuts or cod fish, for example, could have either or both oftwo effects: (1) not imparting an allergic response on their own and (2)conferring protection from the unmodified source by acting as animmunotherapeutic agent for the unmodified source. Methods forengineering of plants and animals are well known and have been for adecade. For example, for plants see Day, (1996) Crit. Rev. Food Sci. &Nut. 36(S), 549-567, the teachings of which are incorporated herein. Seealso Fuchs and Astwood (1996) Food Tech. 83-88. Methods for makingrecombinant animals are also well established. See, for example, Colman,A “Production of therapeutic proteins in the milk of transgeniclivestock” (1998) Biochem. Soc. Symp. 63, 141-147; Espanion and Niemann,(1996) DTW Dtxch Tierarztl Wochenschr 103(8-9), 320-328; and Colman, Am.J. Clin. Nutr. 63(4), 639S-6455S, the teachings of which areincorporated herein. One can also induce site specific changes usinghomologous recombination and/or triplex forming oligomers. See, forexample, Rooney and Moore, (1995) Proc. Natl. Acad. Sci. USA 92,2141-2149; Agrawal, et al., Bio World Today, vol. 9, no. 41, p. 1“Chimeriplasty—Gene Surgery, Not Gene Therapy—Fixes Flawed GenomicSequences” David N. Leff.

Production and Screening of Compounds blocking IgE Binding Sites

Once the IgE binding sites have been identified, it is also possible toblock or limit binding to one or more of these sites by reacting theallergen with a compound that does not prevent the allergen fromactivating T cells, and in some embodiments does not significantly alteror decrease IgG binding capacity, resulting in a modified allergensimilar in functionality to that produced by mutation. There are twoprincipal ways to obtain compounds which block IgE binding sites:combinatorial libraries and combinatorial chemistry.

Identification of Compounds That Mask IgE Binding Sites throughApplication of Combinatorial Chemistry

In some cases it may be preferable to utilize non-peptide compounds toblock binding of IgE to the allergen by masking the IgE binding epitope.This can be accomplished by using molecules that are selected from acomplex mixture of random molecules in what has been referred to as “invitro genetics” or combinatorial chemistry (Szostak, TIBS 19:89, 1992).In this approach a large pool of random and defined sequences issynthesized and then subjected to a selection and enrichment process.The selection and enrichment process involves the binding of the IgEbinding epitopes to a solid support, followed by interaction with theproducts of various combinatorial libraries. Those molecules which donot bind these molecules at all are removed immediately by elution witha suitable solvent. Those molecules which bind to the epitopes willremain bound to the solid support, whereas, unbound compounds will beremoved from the column. Those compounds bound to the column can beremoved, for example, by competitive binding. Following removal of thesecompounds, the compounds which have bound can be identified, usingmethodology well known to those of skill in the art, to isolate andcharacterize those compounds which bind to or interact with IgE bindingepitopes. The relative binding affinities of these compounds can becompared and optimum compounds identified using competitive bindingstudies which are well known to those of skill in the art.

Identification of Compounds That Interact with IgE Binding Sites throughApplication of Combinatorial Phage Display Libraries

Recombinant, monoclonal Fabs directed against conformational epitopes,identified as described above, can be used as reagents to assist in thedefinition of the biochemical nature of these epitopes. Cross-linkingstudies employing derivatized Fabs can be employed to label amino acidresidues in the vicinity of the epitopes. Similarly, the Fabs can beused in protease protection studies to identify those domains of theallergen protein which are shielded from protease degradation bypre-binding of a specific Fab. Experiments employing recombinantmonoclonal Fabs as reagents to label or protect from labeling shouldpermit at least partial elucidation of the structures of conformationalepitopes.

“Humanized” recombinant Fabs should bind to allergens if injected into apatient and thus prevent the binding of these allergens to native IgE.Since the Fabs cannot interact with the Fcε receptor, the binding of theIgE Fabs to allergen would not be expected to elicit mast celldegranulation. Allergen should be neutralized as it is by protectiveIgGs.

Anti-idiotype antibodies directed against the conformationalepitope-specific Fabs should resemble the conformation epitopesthemselves. Injection of these anti-idiotype antibodies should inducethe production of anti-anti-idiotype IgGs which would recognize, bind toand inactivate the conformational epitopes. The method through which theanti-idiotype antibodies would be produced (i.e. animal immunization,“in vitro” immunization or recombinant phage display library) would haveto be determined. Similarly, the possibility that the anti-idiotypeantibodies (which resemble the conformational epitopes) would berecognized by patient IgEs and induce mast cell degranulation needs tobe considered.

II. Diagnostic and Therapeutic Procedures Using Modified Allergens

It is important to administer the modified allergen to an individual(human or animal) to decrease the clinical symptoms of allergic diseaseby using a method, dosage, and carrier which are effective. Allergenwill typically be administered in an appropriate carrier, such as salineor a phosphate saline buffer. Allergen can be administered by injectionsubcutaneously, intramuscularly, or intraperitoneally (most humans wouldbe treated by subcutaneous injection), by aerosol, inhaled powder, or byingestion.

Therapy or desensitization with the modified allergens can be used incombination with other therapies, such as allergen-non-specific anti-IgEantibodies to deplete the patient of allergen-specific IgE antibodies(Boulet, et al. (1997) 155:1835-1840; Fahy, et al. (1997) American JRespir. Crit. Care Med. 155:1828-1834; Demoly, P. and Bousquet (1997) JAm J Resp. Crit. Care Med. 155:1825-1827), or by the pan specificanti-allergy therapy described in U.S. Ser. No. 08/090,375 filed Jun. 4,1998, by M. Caplan and H. Sosin. Therapy with the modified allergen canalso be administered in combination with an adjuvant such as IL 12, IL16, IL 18, Ifn-ζ.

The nucleotide molecule encoding the modified allergen can also beadministered directly to the patient, for example, in a suitableexpression vector such as a plasmid, which is injected directly into themuscle or dermis, or through administration of genetically engineeredcells.

In general, effective dosages will be in the picogram to milligramrange, more typically microgram to milligram. Treatment will typicallybe between twice/weekly and once a month, continuing for up to three tofive years, although this is highly dependent on the individual patientresponse.

The modified allergen can also be used as a diagnostic to characterizethe patient's allergies, using techniques such as those described in theexamples.

EXAMPLES

Peanut allergy is one of the most common and serious of the immediatehypersensitivity reactions to foods in terms of persistence and severityof reaction. Unlike the clinical symptoms of many other food allergies,the reactions to peanuts are rarely outgrown, therefore, most diagnosedchildren will have the disease for a lifetime (Sampson, H. A., andBurks, A. W. (1996) Annu. Rev. Nutr. 16, 161-77; Bock, S. A. (1985) J.Pediatr. 107, 676-680). The majority of cases of fatal food-inducedanaphylaxis involve ingestion of peanuts (Sampson et al., (1992) NEJM327, 380-384; Kaminogawa, S. (1996) Biosci. Biotech. Biochem. 60,1749-1756). The only effective therapeutic option currently availablefor the prevention of a peanut hypersensitivity reaction is foodavoidance. Unfortunately, for a ubiquitous food such as a peanut, thepossibility of an inadvertent ingestion is great.

The examples described below demonstrate identification, modification,and assessment of allergenicity of the major peanut allergens, Ara h 1,Ara h 2, and Ara h 3. Detailed experimental procedures are included forExample 1. These same procedures were used for Examples 2-5. Thenucleotide sequences of Ara h 1, Ara h 2, and Ara h 3, are shown in SEQID NOs. 1, 3, and 5, respectively. The amino acid sequences of Ara h 1,Ara h 2, and Ara h 3 are shown in SEQ ID NOs. 2, 4, and 6 respectively.

Example 1 Identification of Linear IgE Binding Epitopes

Due to the significance of the allergic reaction and the widening use ofpeanuts as protein extenders in processed foods, there is increasinginterest in defining the allergenic proteins and exploring ways todecrease the risk to the peanut-sensitive individual. Various studiesover the last several years have identified the major allergens inpeanuts as belonging to different families of seed storage proteins(Burks, et al. (1997) Eur. J. Biochem. 245, 334-339; Stanley, et al.(1997) Arch. Biochem. Biophys. 342, 244-253). The major peanut allergensAra h 1, Ara h 2, and Ara h 3 belong to the vicilin, conglutin andglycinin families of seed storage proteins, respectively. Theseallergens are abundant proteins found in peanuts and are recognized byserum IgE from greater than 95% of peanut sensitive individuals,indicating that they are the major allergens involved in the clinicaletiology of this disease (Burks, et al. (1995) J. Clinical Invest., 96,1715-1721). The genes encoding Ara h 1 (SEQ ID NO. 1), Ara h 2 (SEQ IDNO. 3), and Ara h 3 (SEQ ID NO. 5) and the proteins encoded by thesegenes (SEQ ID NO. 2, 4, 6) have been isolated and characterized. Thefollowing studies were conducted to identify the IgE epitopes of theseallergens recognized by a population of peanut hypersensitive patientsand a means for modifying their affinity for IgE.

Experimental Procedures

Serum IgE. Serum from 15 patients with documented peanuthypersensitivity reactions (mean age, 25 yrs) was used to determinerelative binding affinities between wild-type and mutant Ara h 1synthesized epitopes. The patients had either a positive double-blind,placebo-controlled, food challenge or a convincing history of peanutanaphylaxis (laryngeal edema, severe wheezing, and/or hypotension;Burks, et al. (1988) J. Pediatr. 113, 447-451). At least 5 ml of venousblood was drawn from each patient, allowed to clot, and serum wascollected. A serum pool from 12 to 15 patients was made by mixing equalaliquots of serum IgE from each patient. The pools were then used inimmunoblot analysis.

Peptide synthesis. Individual peptides were synthesized on a derivatizedcellulose membrane using 9-fluorenyllmethoxycarbonyl (Fmoc) amino acidactive esters according to the manufacturer's instructions (GenosysBiotechnologies, Woodlands, Tex.; Fields, G. B. and Noble, R. L. (1990)Int. J. Peptide Protein Res. 35, 161-214). Fmoc-amino acids (N-terminalblocked) with protected side chains were coupled in the presence of1-methyl-2-pyrrolidone to a derivatized cellulose membrane. Followingwashing with dimethylformamide (DMF), unreacted terminal amino groupswere blocked from further reactions by acetylation with aceticanhydride. The N-terminal Fmoc blocking group was then removed byreaction with 20% piperidine and 80% DMF, v/v. The membrane was washedin DMF followed by methanol, the next reactive Fmoc-amino acid was thencoupled as before, and the sequence of reactions was repeated with thenext amino acid. When peptide synthesis was complete, the side chainswere deprotected with a mixture of dichloromethane (DCM),triflouroacetic acid, and triisobutylsilane (1.0:1.0:0.5), followed bysuccessive washes in DCM, DMF, and methanol. Peptides synthesisreactions were monitored by bromophenol blue color reactions duringcertain steps of synthesis. Cellulose derivitised membranes andFmoc-amino acids were supplied by Genosys Biotechnologies. All otherchemical were purchased from Aldrich Chemical Company, Inc. (Milwaukee,Wis.) or Fluka (Bucks, Switzerland). Membranes were either probedimmediately or stored at −20° C. until needed.

IgE binding assays. Cellulose membranes containing synthesized peptideswere washed 3 times in Tris-buffered saline (TBS; 136 mM NaCl, 2.7 mMKCl, and 50 mM trizma base pH 8.0) for 10 min at room temperature (RT)and then incubated overnight in blocking buffer: [TBS, 0.05% Tween™ 20;concentrated membrane blocking buffer supplied by Genosys; and sucrose(0.0:1.0:0.5)]. The membrane was then incubated in pooled sera dilutedin 1:5 in 20 mM Tris-Cl pH 7.5, 150 mM NaCl, and 1 % bovine serumalbumin overnight at 4° C. Primary antibody was detected with¹²⁵I-labeled equine anti-human IgE (Kallestad, Chaska, Minn.).

Quantitation of IgE binding. Relative amounts of IgE binding toindividual peptides were determined by a Bio-Rad (Hercules, CA) modelGS-700 imaging laser densitometer and quantitated with Bio-Rad molecularanalyst software. A background area was scanned and subtracted from theobtained values. Following quantitation, wild-type intensities werenormalized to a value of one and the mutants were calculated aspercentages relative to the wild-type.

Synthesis and purification of recombinant Ara h 2 protein. cDNA encodingAra h 2 was placed in the pET-24b expression vector. The pET-24expression vector places a 6×histidine tag at the carboxyl end of theinserted protein. The histidine tag allows the recombinant protein to bepurified by affinity purification on a nickel column (HisBind resin).Recombinant Ara h 2 was expressed and purified according to theinstructions of the pET system manual. Briefly, expression of therecombinant Ara h 2 was induced in 200 ml cultures of strain BL21(DE3)E. coli with 1 mM IPTG, at mid log phase. Cultures were allowed tocontinue for an additional 3 hours at 36° C. Cells were harvested bycentrifugation at 2000×g for 15 minutes and then lysed in denaturingbinding buffer (6 M urea, 5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH7.9). Lysates were cleared by centrifugation at 39,000×g for 20 minutesfollowed by filtration though 0.45 micron filters. The cleared lysatewas applied to a 10 ml column of HisBind resin, washed with imidazolewash buffer (20 mM imidazole, 6 M urea, 0.5 M NaCl, 20 mM Tris-HCl, pH7.9). The recombinant Ara h 2 was then released from the column usingelution buffer (1 M imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). Theelution buffer was replaced with phosphate buffered saline by dialysis.The purification of recombinant Ara h 2 was followed by SDS PAGE andimmunoblots. Peanut specific serum IgE was used as a primary antibody.

Skin prick tests. The ability of purified native and recombinant Ara h 2to elicit the IgE mediated degranulation of mast cells was evaluatedusing prick skin tests in a peanut allergic individual. An individualmeeting the criteria for peanut allergy (convincing history or positivedouble blind placebo controlled food challenge) and a non-allergiccontrol were selected for the testing. Purified native and recombinantAra h 2 and whole peanut extract (Greer Laboratories, Lenoir, N.C.) weretested. Twenty microliters of the test solution were applied to theforearm of the volunteer and the skin beneath pricked with a sterileneedle. Testing was started at the lowest concentration (less than orequal to 1 mg/ml) and increased ten fold each round to the highestconcentration or until a positive reaction was observed. Mean diametersof the wheal and erythema were measured and compared to the negativesaline control. A positive reaction was defined as a wheal 3 mm largerthen the negative control. Histamine was used as the positive control.

Results

Identification of the linear IgE-binding epitopes of Ara h 1, Ara h 2and Ara h 3 allergens. Epitope mapping was performed on the Ara h 1, Arah 2 and Ara h 3 allergens by synthesizing each of these proteins in 15amino acid long overlapping peptides that were offset from each other by8 amino acids. The peptides were then probed with a pool of serum IgEfrom 15 patients with documented peanut hypersensitivity. This analysisresulted in multiple IgE binding regions being identified for eachallergen. The exact position of each IgE binding epitope was thendetermined by re-synthesizing these IgE reactive regions as 10 or 15amino acid long peptides that were offset from each other by two aminoacids. These peptides were probed with the same pool of serum IgE frompeanut sensitive patients as used before. An example of this procedurefor each of the peanut allergens is shown in FIG. 1-3 (Ara h 1—FIG. 1;Ara h 2—FIG. 2; Ara h 3—FIG. 3). This analysis revealed that there were23 linear IgE binding epitopes on Ara h 1, 10 epitopes on Ara h 2, and 4epitopes on Ara h 3.

In an effort to determine which, if any, of the epitopes were recognizedby the majority of patients with peanut hypersensitivity, each set ofepitopes identified for the peanut allergens were synthesized and thenprobed individually with serum IgE from 10 different patients. All ofthe patient sera tested recognized multiple epitopes.

Table 1 shows the amino acid sequence and position of each epitopewithin the Ara h 1 protein of all 23 IgE binding epitopes mapped to thismolecule. Table 2 shows the amino acid sequence and position of eachepitope within the Ara h 2 protein of all 10 IgE binding epitopes mappedto this molecule. Table 3 shows the amino acid sequence and position ofeach epitope within the Ara h 3 protein of all 4 IgE binding epitopesmapped to this molecule.

Four epitopes of the Ara h 1 allergen (peptides 1, 3, 4, 17 of Table 1),three epitopes of the Ara h 2 allergen (peptides 3, 6, 7 of Table 2),and 1 epitope of the Ara h 3 allergen (peptide 2 of Table 3) wereimmunodominant. TABLE 1 Ara h 1 IgE Binding Epitopes EPITOPE AA SEQUENCEPOSITION SEQ ID NO. 1 AKSSPYOKKT 25-34 7 2 QEPDDLKQKA 48-57 8 3LEYDPRLVYD 65-74 9 4 GERTRGRQPG 89-98 10 5 PGDYDDDRRQ  97-106 11 6PRREEGGRWG 107-116 12 7 REREEDWRQP 123-132 13 8 EDWRRPSHQQ 134-143 14 9QPRKIRPEGR 143-152 15 10 TPGQFEDFFP 294-303 16 11 SYLQEFSRNT 311-320 1712 FNAEFNEIRR 325-334 18 13 EQEERGQRRW 344-353 19 14 DITNPINLRE 393-40220 15 NNFGKLFEVK 409-418 21 16 GTGNLELVAV 461-470 22 17 RRYTARLKEG498-507 23 18 ELHLLGFGIN 525-534 24 19 HRIFLAGDKD 539-548 25 20IDQIEKQAKD 551-560 26 21 KDLAFPGSGE 559-568 27 22 KESHFVSARP 578-587 2823 PEKESPEKED 597-606 29

The underlined portions of each peptide are the smallest IgE bindingsequences as determined by this analysis. All of these sequences can befound in SEQ ID NO 2. TABLE 2 Ara h 2 IgE Binding Epitopes EPITOPE AASEQUENCE POSITION SEQ ID NO. 1 HASARQQWEL 15-24 30 2 QWELQGDRRC 21-30 313 DRRCQSQLER 27-36 32 4 LRPCEQHLMQ 39-48 33 5 KIQRDEDSYE 49-58 34 6YERDPYSPSQ 57-66 35 7 SQDPYSPSPY 65-74 36 8 DRLQGRQQEQ 115-124 37 9KRELRNLPQQ 127-136 38 10 QRCDLDVESG 143-152 39

The underlined portions of each peptide are the smallest IgE bindingsequences as determined by this analysis. All of these sequences can befound in SEQ ID NO 4. TABLE 3 Ara H3 I-E Binding Epitopes EPITOPE AASEQUENCE POSITION SEQ ID NO. 1 IETWNPNNQEFECAG 33-47 40 2GNIFSGFTPEFLEQA 240-254 41 3 VTVRGGLRILSPDRK 279-293 42 4DEDEYEYDEEDRRRG 303-317 43

The underlined portions of each peptide are the smallest IgE bindingsequences as determined by this analysis. All of these sequences can befound in SEQ ID NO 6.

Example 2 Modification of Peanut Allergens to Decrease Allergenicity

The major linear IgE binding epitopes of the peanut allergens weremapped using overlapping peptides synthesized on an activated cellulosemembrane and pooled serum IgE from 15 peanut sensitive patients, asdescribed in Example 1. The size of the epitopes ranged from six tofifteen amino acids in length. The amino acids essential to IgE bindingin each of the epitopes were determined by synthesizing duplicatepeptides with single amino acid changes at each position. These peptideswere then probed with pooled serum IgE from 15 patients with peanuthypersensitivity to determine if the changes affected peanut-specificIgE binding. For example, epitope 9 in Table 1 was synthesized with analanine or methionine residue substituted for one of the amino acids andprobed. The following amino acids were substituted (first letter is theone-letter amino acid code for the residue normally at the position, theresidue number, followed by the amino acid that was substituted for thisresidue; the numbers indicate the position of each residue in the Ara h1 protein, SEQ ID NO. 2): Q143A, P144A; R145A; KI46A; I147A; R148A;PI49A; E150A; G151A; R152A; Q143M; P144M; R145M; K146M; I147M; R148M;P149M; E150M; G151M; R152M. The immunoblot strip containing thewild-type and mutated peptides of epitope 9 showed that binding ofpooled serum IgE to individual peptides was dramatically reduced wheneither alanine or methionine was substituted for each of the amino acidsat positions 144, 145, and 147-150 of Ara h 1 shown in SEQ ID NO. 2.Changes at positions 144, 145, 147, and 148 of Ara h 1 shown in SEQ IDNO. 2 had the most dramatic effect when methionine was substituted forthe wild-type amino acid, resulting in less than 1% of peanut specificIgE binding to these peptides. In contrast, the substitution of analanine for arginine at position 152 of Ara h 1 shown in SEQ ID NO. 2resulted in increased IgE binding. The remaining Ara h 1 epitopes, andthe Ara h 2 and Ara h 3 epitopes, were tested in the same manner and theintensity of IgE binding to each spot was determined as a percentage ofIgE binding to the wild-type peptide. Any amino acid substitution thatresulted in less than 1% of IgE binding when compared to the wild-typepeptide was noted and is indicated in Tables 4-6. Table 4 shows theamino acids that were determined to be critical to IgE binding in eachof the Ara h 1 epitopes. Table 5 shows the amino acids that weredetermined to be critical to IgE binding in each of the Ara h 2epitopes. Table 6 shows the amino acids that were determined to becritical to IgE binding in each of the Ara h 3 epitopes. This analysisindicated that each epitope could be mutated to a non-IgEbinding-peptide by the substitution of a single amino acid residue.

The results discussed above for Ara h 1, Ara h 2, and Ara h 3demonstrate that once an IgE binding site has been identified, it ispossible to reduce IgE binding to this site by altering a single aminoacid of the epitope. The observation that alteration of a single aminoacid leads to the loss of IgE binding in a population ofpeanut-sensitive individuals is significant because it suggests thatwhile each patient may display a polyclonal IgE reaction to a particularallergen, IgE from different patients that recognize the same epitopemust interact with that epitope in a similar fashion. Besides findingthat many epitopes contained more than one residue critical for IgEbinding, it was also determined that more than one residue type (ala ormet) could be substituted at certain positions in an epitope withsimilar results. This allows for the design of a hypoallergenic proteinthat would be effective at blunting allergic reactions for a populationof peanut sensitive dividuals. Furthermore, the creation of a plantproducing a peanut where the IgE binding epitopes of the major allergenshave been removed should prevent the development of peanuthypersensitivity in individuals genetically predisposed to this foodallergy. TABLE 4 Amino Acids Critical to IgE Binding of Ara h 1 EPITOPEAA SEQUENCE POSITION SEQ ID NO. 1 AKS SPY Q K KT 25-34 7 2 QEP DDL KQKA48-57 8 3 LE YDP RL VY D 65-74 9 4 GE R TR GRQ PG 89-98 10 5 PGDYDD DRRQ  97-106 11 6 PRREE G GRWG 107-116 12 7 REREED W R Q P 123-132 13 8EDW RRP SHQQ 134-143 14 9 Q PR K IR PEGR 143-152 15 10 T P GQ F ED FF P294-303 16 11 S YL Q EF SRNT 311-320 17 12 FNAE F NEIRR 325-334 18 13EQEER G QRRW 344-353 19 14 DIT NPI N L RE 393-402 20 15 NNFGK LF EVK409-418 21 17 RRY TARLKEG 498-507 23 18 EL HL L GFG IN 525-534 24 19HRIFLAGD K D 539-548 25 20 IDQ I EKQ A K D 551-560 26 21 KDLA FPG SGE559-568 27 22 KESHFV S ARP 578-587 28Note.The Ara h 1 IgE binding epitopes are indicated as the single letteramino acid code. The position of each peptide with respect to the Ara h1 protein is indicated in the right hand column. The amino acids that,when altered, lead to loss of IgE binding are shown as the bold,underlined residues. Epitopes 16 and 23 were not included in this studybecause they were recognized by a single patient who was no longeravailable to the study. All of these sequences can be found in SEQ ID NO2.

TABLE 5 Amino Acids Critical to IgE Binding of Ara h 2 EPITOPE AASEQUENCE POSITION SEQ ID NO. 1 HASAR Q Q W EL 15-24 30 2 Q W E L Q GDRRC 21-30 31 3 D RR C Q SQL ER 27-36 32 4 L R P CE QH LMQ 39-48 33 5 KIQ RD E D SYE 49-58 34 6 YER DPY SPSQ 57-66 35 7 SQ DPY SPSPY 65-74 36 8DRL QGR QQEQ 115-124 37 9 KR E L RN L PQQ 127-136 38 10 QRC DL D VE SG143-152 39Note.The Ara h 2 IgE binding epitopes are indicated as the single letteramino acid code. The position of each peptide with respect to the Ara h2 protein is indicated in the right hand column. The amino acids that,when altered, lead to loss of IgE binding are shown as the bold,underlined residues. All of these sequences can be found in SEQ ID NO 4.

TABLE 6 Amino Acids Critical to IgE-Binding of Ara h 3. EPITOPE AASEQUENCE POSITION SEQ ID NO. 1 IETWN PN NQEFECAG 33-47 40 2 GNI F SG FTPE FL EQA 240-254 41 3 VTVRGG L R IL S P DRK 279-293 42 4 DEDEY EYDE EDR RRG 303-317 43Note.The Ara h 3 IgE binding epitopes are indicated as the single letteramino acid code. The position of each peptide with respect to the Ara h3 protein is indicated in the right hand column. The amino acids that,when altered, lead to loss of IgE binding are shown as the bold,underlined. All of these sequences can be found in SEQ ID NO 6.

Example 3 A Modified Ara h 2 Protein Binds less IgE But Similar Amountsof IgG

In order to determine the effect of changes to multiple epitopes withinthe context of the intact allergen, four epitopes (including the threeimmunodominant epitopes) of the Ara h 2 allergen were mutagenized andthe protein produced recombinantly. The amino acids at position 20, 31,60, and 67 of the Ara h 2 protein (shown in SEQ ID NO. 4 and FIG. 4A)were changed to alanine by mutagenizing the gene encoding this proteinby standard techniques. These residues are located in epitopes 1, 3, 6,and 7 and represent amino acids critical to IgE binding that weredetermined in Example 2. The modified and wild-type versions of thisprotein were produced and immunoblot analysis performed using serum frompeanut sensitive patients. These results showed that the modifiedversion of this allergen bound significantly less IgE than the wild-typeversion of these recombinant proteins (FIG. 4B) but bound similaramounts of IgG.

Example 4 A Modified Ara h 2 Protein Retains the Ability to StimulateT-cells to Proliferate

The modified recombinant Ara h 2 protein described in Example 3 was usedin T-cell proliferation assays to determine if it retained the abilityto activate T cells from peanut sensitive individuals. Proliferationassays were performed on T-cell lines grown in short-term culturedeveloped from six peanut sensitive patients. T-cells lines werestimulated with either 50 μg of crude peanut extract, 10 μg of nativeAra h 2, 10 μg of recombinant wild-type Ara h 2, or 10 μg of modifiedrecombinant Ara h 2 protein and the amount of 3H-thymidine determinedfor each cell line. Results were expressed as the average stimulationindex (SI) which reflected the fold increase in 3H-thymidineincorporation exhibited by cells challenged with allergen when comparedwith media treated controls (FIG. 5).

Example 5 A Modified Ara h 2 Protein Elicits a Smaller Wheal and Flarein Skin Prick Tests of a Peanut Sensitive Individual

The modified recombinant Ara h 2 protein described in Example 3 and thewild-type version of this recombinant protein were used in a skin pricktest of a peanut sensitive individual. Ten micrograms of these proteinswere applied separately to the forearm of a peanut sensitive individual,the skin pricked with a sterile needle, and 10 minutes later any whealand flare that developed was measured. The wheal and flare produced bythe wild-type Ara h 2 protein (8 mm×7 mm) was approximately twice aslarge as that produced by the modified Ara h 2 protein (4 mm×3 mm). Acontrol subject (no peanut hypersensitivity) tested with the sameproteins had no visible wheal and flare but, as expected, gave positiveresults when challenged with histamine. In addition, the test subjectgave no positive results when tested with PBS alone. These resultsindicate that an allergen with only 40% of its IgE binding epitopesmodified (4/10) can give measurable reduction in reactivity in an invivo test of a peanut sensitive patient.

These same techniques can be used with the other known peanut allergens,Ara h 1 (SEQ ID NO 1 and 2), Ara h 3 (SEQ ID NO. 5 and 6), or any otherallergen.

Modifications and variations of the methods and materials describedherein will be obvious to those skilled in the art. Such modificationsand variations are intended to come within the scope of the appendedclaims.

1. A modified protein allergen whose amino acid sequence issubstantially identical to that of an unmodified protein allergen exceptthat at least one amino acid has been modified in at least one IgEepitope so that IgE binding to the modified protein allergen is reducedas compared with IgE binding to the unmodified protein allergen, the atleast one IgE epitope being one that is recognized when the unmodifiedprotein allergen is contacted with serum IgE from an individual that isallergic to the unmodified protein allergen.
 2. The modified proteinallergen of claim 1 wherein at least one amino acid has been modified inall the IgE epitopes of the unmodified protein allergen.
 3. The modifiedprotein allergen of claim 1 wherein the at least one IgE epitope is onethat is recognized when the unmodified protein allergen is contactedwith a pool of sera IgE taken from a group of at least two individualsthat are allergic to the unmodified protein allergen.
 4. The modifiedprotein allergen of claim 1 wherein at least one modified amino acid islocated in the center of the at least one IgE epitope.
 5. The modifiedprotein allergen of claim 1 wherein at least one amino acid in the atleast one IgE epitope of the unmodified protein allergen has beenmodified by substitution.
 6. The modified protein allergen of claim 5wherein at least one hydrophobic amino acid in the at least one IgEepitope of the unmodified protein allergen has been substituted by aneutral or hydrophilic amino acid.
 7. The modified protein allergen ofclaim 1 wherein the modified protein allergen retains the ability toactivate T cells.
 8. The modified protein allergen of claim 1 whereinthe modified protein allergen retains the ability to bind IgG.
 9. Themodified protein allergen of claim 1 wherein the modified proteinallergen retains the ability to initiate a Th1-type response.
 10. Themodified protein allergen of claim 1 wherein the modified proteinallergen is a portion of the unmodified protein allergen.
 11. Acomposition comprising the modified protein allergen of claim 1 and anadjuvant selected from the group consisting of IL-12, IL-16, IL-18,IFNγ, and immune stimulatory sequences.
 12. The modified proteinallergen of claim 1 wherein the modified protein allergen is made in atransgenic plant or animal.
 13. The modified protein allergen of claim 1expressed in a recombinant host selected from the group consisting ofplants and animals.
 14. The modified protein allergen of claim 1expressed in a recombinant host selected from the group consisting ofbacteria, yeast, fungi, and insect cells.
 15. The modified proteinallergen of claim 1 wherein the unmodified protein allergen is obtainedfrom a source selected from the group consisting of legumes, milks,grains, eggs, fish, crustaceans, mollusks, insects, molds, dust,grasses, trees, weeds, mammals, and natural latexes.
 16. The modifiedprotein allergen of claim 1 made by the process of: identifying at leastone IgE epitope in an unmodified protein allergen; preparing at leastone modified protein allergen whose amino acid sequence is substantiallyidentical to that of the unmodified protein allergen except, that atleast one amino acid has been modified in the at least one IgE epitope;screening for IgE binding to the at least one modified protein allergensby contacting the at least one modified protein allergens with serum IgEtaken from at least one individual that is allergic to the unmodifiedprotein allergen; and selecting a modified protein allergen withdecreased binding to IgE as compared to the unmodified protein allergen.17. A modified food allergen whose amino acid sequence is substantiallyidentical to that of an unmodified food allergen except that at leastone amino acid has been modified in at least one IgE epitope so that IgEbinding to the modified food allergen is reduced as compared with IgEbinding to the unmodified food allergen, the at least one IgE epitopebeing one that is recognized when the unmodified food allergen iscontacted with serum IgE from an individual that is allergic to theunmodified food allergen.
 18. The modified food allergen of claim 17wherein the unmodified food allergen is obtained from a source selectedfrom the group consisting of legumes, milks, grains, eggs, fish,crustaceans, and mollusks.
 19. The modified food allergen of claim 18wherein the unmodified food allergen is obtained from a source selectedfrom the group consisting of wheat, barley, cow milk, egg, codfish,hazel nut, soybean, and shrimp.
 20. A modified peanut allergen whoseamino acid sequence is substantially identical to that of an unmodifiedpeanut allergen except that at least one amino acid has been modified inat least one IgE epitope so that IgE binding to the modified peanutallergen is reduced as compared with IgE binding to the unmodifiedpeanut allergen, the at least one IgE epitope being one that isrecognized when the unmodified peanut allergen is contacted with serumIgE from an individual that is allergic to the unmodified peanutallergen.
 21. The modified peanut allergen of claim 20 wherein theunmodified peanut allergen is selected from the group consisting of Arah 1, Ara h 2, and Ara h
 3. 22. The modified allergen of claim 1, claim17, or claim 20, wherein the at least one IgE epitope contains 1-6 aminoacid residues that are modified as compared with the unmodifiedallergen.
 23. The modified allergen of claim 1, claim 17, or claim 20,wherein the at least one IgE epitope contains 1-5 amino acid residuesthat are modified as compared with the unmodified allergen.
 24. Themodified allergen of claim 1, claim 17, or claim 20, wherein the atleast one IgE epitope contains 1-4 amino acid residues that are modifiedas compared with the unmodified allergen.
 25. The modified allergen ofclaim 1, claim 17, or claim 20, wherein the at least one IgE epitopecontains 1-3 amino acid residues that are modified as compared with theunmodified allergen.
 26. The modified allergen of claim 1, claim 17, orclaim 20, wherein the at least one IgE epitope contains 1-2 amino acidresidues that are modified as compared with the unmodified allergen. 27.The modified allergen of claim 1, claim 17, or claim 20, wherein the atleast one IgE epitope contains 1 amino acid residue that is modified ascompared with the unmodified allergen.
 28. The modified allergen ofclaim 1, claim 17, or claim 20, wherein binding by serum IgE to the atleast one epitope is reduced for the modified allergen to less thanabout 1% of that observed to the unmodified allergen.